*NCBI Gene # or RefSeq#: *Protein ID (NP or XP #) or Wolbachia#: *Organism (including strain):Vittaforma corneae Etiologic Risk Group (see link below): *Background/Disease Information (sort of like the Intro to your Mini Research Write up): Vittaforma corneae is a human infecting microsporidia. It causes eye infections in immunocompetent people and infections in immunocompromised AIDS patients. Microsporidia can infect almost all species on earth. This specific organism can cause keratitis and systemic infections. Infection is determined through testing urine and other bodily fluids for the spores. Most current treatments do not affect the organism significantly so there is a great need for new drugs and treatments. Failure of adequate treatment results in blindness in afflicted individuals. The MetAP2 enzyme playes a role in tissue repair and possibly cell growth .
Essentiality of this protein:
MetAp2 is a part of the methionyl aminopeptidase family associated with protein coding. When the protein is inhibited, a common reaction is that the cell eventually dies. When the protein is over expressed, the result can either be cell death or over growth of cells resulting in various forms of cancer. http://www.genecards.org/cgi-bin/carddisp.pl?gene=METAP2
Complex of proteins?: Methionyl Aminopeptidase Family
-- List cost and quantity of substrate reagents and supplier -DTNB -- $22
Structure Available (PDB or Homology model)
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage: 95%
---- Max % Identities: 45%
---- % Positives: 63%
Current Inhibitors: Fumagillin, Ovalicin
Expression Information (has it been expressed in bacterial cells):
It has been expressed inhuman, rabbit, and Encephalitozoon cunicul.
Purification Method:
Microsporidian spores were purified by filtration through nucleopore filters, treated with 0.2% SDS for 20 min, washed with PBS and resuspended in proteinase K buffer [10 mM pH 7.5 Tris HCl, 10 mM EDTA, 150 mM NaCl, 0.4% SDS]. Purified spores were then disrupted using acid-washed 500-μm glass beads in a Mini-Bead beater (Bio-spec Products Inc., Bartlesville, OK). Proteinase K was added to the disrupted spores and the solution was incubated for 15 min at 65°C. DNA was then prepared by phenol/chloroform extraction followed by ethanol precipitation.
Image of protein (PyMol with features delineated and shown separately):
http://www.rcsb.org/pdb/images/1b59_bio_r_500.jpg
1B59- Human MetAp2 Complexed with Ovalicin
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): MSFVLLNKIEPQPIEFLEPASFNLTSDSILNDALRAAEAHRRVRSSVQQILKPGVSLLQIIETVEKATRA LLVGEKNNGIGFPCGVSLNDCAAHFTLNPGDRNIILKESDILKIDFGTHSNGRIMDSAFTVCFDPKFEQL LKASQEATKRGLEVIGIDMMVCEIGREISEVFKSFEIELDNKIMPIKPIWNLNGHSIEQYRIHGEFSIPP INNGDTSRVSEGFCAIETFASTGKGEVCDKGEASHFMINRNPPSSKIYNAKNEKVLKTIQKEFGTLPFSP RHVEFYSPDSFSSIKLLSLRKFIDPYPPLHDLPNSFVAQFEHTVYLSDTGKRILTNGNDY
*length of your protein in Amino Acids: 340
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 79kDa
Molar Extinction coefficient of your protein at 280 nm wavelength:
14815
*GC% Content for gene: *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
Target : Methionyl Aminopeptidase 2 (MetAP2)
*NCBI Gene # or RefSeq#:*Protein ID (NP or XP #) or Wolbachia#:
*Organism (including strain): Vittaforma corneae
Etiologic Risk Group (see link below):
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Vittaforma corneae is a human infecting microsporidia. It causes eye infections in immunocompetent people and infections in immunocompromised AIDS patients. Microsporidia can infect almost all species on earth. This specific organism can cause keratitis and systemic infections. Infection is determined through testing urine and other bodily fluids for the spores. Most current treatments do not affect the organism significantly so there is a great need for new drugs and treatments. Failure of adequate treatment results in blindness in afflicted individuals. The MetAP2 enzyme playes a role in tissue repair and possibly cell growth .
Essentiality of this protein:
MetAp2 is a part of the methionyl aminopeptidase family associated with protein coding. When the protein is inhibited, a common reaction is that the cell eventually dies. When the protein is over expressed, the result can either be cell death or over growth of cells resulting in various forms of cancer.
http://www.genecards.org/cgi-bin/carddisp.pl?gene=METAP2
Complex of proteins?: Methionyl Aminopeptidase Family
Druggable Target:
peptide + H2O = N-terminal amino acid + peptide, preferentially methionine, further reaction: arylamide + H2O = aromatic hydrocarbon + amine
EC#: 3.4.11.18
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.4.11.18
Enzyme Assay information (spectro
photometric, coupled assay ?, reagents):
The enzymatic reaction catalyzed by MetAP can be continuously
monitored in real time on a UV-VIS spectrophotometer
by simply adding excess DTNB into a MetAP reaction
mixture. MetAPs of both bacterial and eukaryotic origins
exhibit strong sequence specificity at the N-terminus,
with methionine being the most preferred amino
acid at the P1 position (3, 5, 9, 10, 24–26). At the P19
position, MetAP prefers amino acids with small side
chains (e.g., Gly, Ala, Ser, Thr, Val, Pro, and Cys).
MetAP also requires at least a tripeptide for efficient
hydrolysis, although the identity of the side chains at
P29 position and beyond does not appears to be critical
(9, 10).
http://www.sciencedirect.com/science/article/pii/S0003269700945135
Spectrophotometric assay used a custom synthesized enzyme.
-- link to Sigma (or other company) page for assay or assay reagents (substrates)
http://www.sigmaaldrich.com/catalog/product/aldrich/d218200?lang=en®ion=US
-- link (or citation) to paper that contains assay information
http://www.sciencedirect.com/science/article/pii/S0003269700945135
-- List cost and quantity of substrate reagents and supplier
-DTNB -- $22
Structure Available (PDB or Homology model)
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage: 95%
---- Max % Identities: 45%
---- % Positives: 63%
Current Inhibitors: Fumagillin, Ovalicin
Expression Information (has it been expressed in bacterial cells):
It has been expressed inhuman, rabbit, and Encephalitozoon cunicul.
Purification Method:
Microsporidian spores were purified by filtration through nucleopore filters, treated with 0.2% SDS for 20 min, washed with PBS and resuspended in proteinase K buffer [10 mM pH 7.5 Tris HCl, 10 mM EDTA, 150 mM NaCl, 0.4% SDS]. Purified spores were then disrupted using acid-washed 500-μm glass beads in a Mini-Bead beater (Bio-spec Products Inc., Bartlesville, OK). Proteinase K was added to the disrupted spores and the solution was incubated for 15 min at 65°C. DNA was then prepared by phenol/chloroform extraction followed by ethanol precipitation.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3109671/
Image of protein (PyMol with features delineated and shown separately):
1B59- Human MetAp2 Complexed with Ovalicin
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MSFVLLNKIEPQPIEFLEPASFNLTSDSILNDALRAAEAHRRVRSSVQQILKPGVSLLQIIETVEKATRA LLVGEKNNGIGFPCGVSLNDCAAHFTLNPGDRNIILKESDILKIDFGTHSNGRIMDSAFTVCFDPKFEQL LKASQEATKRGLEVIGIDMMVCEIGREISEVFKSFEIELDNKIMPIKPIWNLNGHSIEQYRIHGEFSIPP INNGDTSRVSEGFCAIETFASTGKGEVCDKGEASHFMINRNPPSSKIYNAKNEKVLKTIQKEFGTLPFSP RHVEFYSPDSFSSIKLLSLRKFIDPYPPLHDLPNSFVAQFEHTVYLSDTGKRILTNGNDY
*length of your protein in Amino Acids: 340
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 79kDa
Molar Extinction coefficient of your protein at 280 nm wavelength:
14815
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
ATGTCCTTTGTACTTCTTAACAAAATAGAGCCTCAACCAATTGAATTTCTGGAACCAGCTTCATTTAATT TAACAAGCGATTCAATATTAAATGACGCCTTACGTGCTGCAGAGGCCCATAGGCGGGTAAGGAGCAGCGT CCAACAGATTCTAAAACCAGGAGTTTCACTACTGCAAATTATAGAAACTGTGGAAAAAGCTACACGAGCT TTACTAGTAGGAGAGAAGAACAACGGAATTGGATTTCCATGTGGTGTAAGCTTAAATGACTGTGCTGCTC ACTTTACACTCAATCCAGGAGATAGGAACATAATATTAAAAGAATCTGATATTCTAAAAATAGATTTTGG AACACATTCTAATGGCAGAATTATGGATTCTGCATTTACAGTCTGTTTTGATCCAAAGTTTGAACAGTTA CTAAAGGCCTCACAAGAAGCTACCAAAAGAGGACTAGAGGTTATAGGAATTGATATGATGGTGTGTGAAA TAGGTAGGGAAATTTCTGAAGTATTCAAATCCTTTGAAATAGAGTTAGATAATAAAATCATGCCGATAAA GCCCATATGGAATCTTAACGGTCATTCTATTGAGCAATATAGAATACATGGTGAGTTTTCAATACCACCA ATCAATAATGGAGACACATCAAGAGTTTCAGAGGGATTCTGTGCCATTGAAACATTTGCAAGTACAGGAA AGGGTGAGGTCTGTGATAAAGGTGAAGCATCCCATTTTATGATCAATAGAAACCCACCAAGTTCAAAAAT TTATAATGCTAAAAATGAAAAAGTTCTTAAGACTATTCAAAAAGAGTTTGGAACATTGCCCTTTAGTCCT AGACATGTTGAGTTTTATAGCCCTGATTCATTTTCAAGTATCAAGCTGCTATCACTTAGAAAATTTATCG ACCCATACCCACCTCTTCATGATTTACCAAACTCCTTTGTTGCACAATTTGAGCATACAGTTTATCTTTC AGACACAGGGAAGAGAATACTTACAAACGGCAACGATTATTAA
*GC% Content for gene:
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
Resources:
http://www.ncbi.nlm.nih.gov/protein/429961911?report=genpepthttp://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.4.11.1
http://www.ncbi.nlm.nih.gov/pubmed/12425527
http://aac.asm.org/content/52/2/790.full
http://pubmedcentralcanada.ca/pmcc/articles/PMC2759695/
See ProtocolTargetDiscoveryVDS.docx for more
Etiologic Risk Group Categories (for pathogens): http://www.utexas.edu/research/rsc/ibc/agent_class.html#_Toc7238334
Databases of genes/organisms:
http://www.niaid.nih.gov/Pages/default.aspx
http://eupathdb.org/eupathdb/
https://patricbrc.vbi.vt.edu/portal/portal/patric/Home
http://www.nmpdr.org/FIG/wiki/view.cgi/Main/EssentialGenes
http://tubic.tju.edu.cn/deg/
http://csgid.org/csgid/cake/pages/community_request_gateway
http://tdrtargets.org/
http://gsc.jcvi.org/status.shtml
Scientific Nomenclature page from Center for Disease Control (gene, protein names and abbreviations)
http://wwwnc.cdc.gov/eid/pages/scientific-nomenclature.htm
Gene Information:
NCBI GENE Page: http://www.ncbi.nlm.nih.gov/gene
BLAST Page: http://blast.ncbi.nlm.nih.gov/
Protein Information:
NCBI Protein Page: http://www.ncbi.nlm.nih.gov/protein
Protein Expression Website
Protein Expression Paper: SGC_ProteinProductionPurificationNatMethods2008.pdf
Primer Overlap PCR Articles
HooverLubkowski_PCRoverlapcloninggnf042.pdf
StemmerPCRoverlapGene1995.pdf
Is my target good for Virtual Screening programs?
Reynolds_THermodynamicsLigandBinding_MedChemLett2011.pdf